Amino acid composition and solubility of proteins isolated from ...

International Food Research Journal 20(6): 2995-3000 (2013) Journal homepage: http://www.ifrj.upm.edu.my

Amino acid composition and solubility of proteins isolated from sunflower meal produced in Bulgaria Ivanova, P., *Chalova, V., Koleva, L. and Pishtiyski, I. University of Food Technologies, Department of Biochemistry and Molecular Biology, Plovdiv-4002, Bulgaria Article history

Abstract

Received: 11 June 2013 Received in revised form: 6 July 2013 Accepted: 12 July 2013

Two protein isolates (PI1 and PI2) were prepared from sunflower meal produced in Bulgaria. They were obtained by using isoelectric or ethanol precipitation following the extraction of the proteins with 10% NaCl. Their amino acid composition and water solubility as a function of pH and presence of NaCl were investigated. Lysine was established as the first limiting amino acid in the protein isolates with amino acid scores of 35.81% (PI1) and 43.09% (PI2). Both protein isolates contained relatively high amounts of sulfur-containing amino acids with amino acid scores of 99.14%. Arginine reached 8.45 and 9.75 g/100 g protein in PI2 and PI1 respectively. Being obtained by isoelectric precipitation, PI1 was highly soluble in either acidic (pH ≤ 3.5) or alkaline (pH ≥ 7) medium. In contrast, PI2 was insoluble for each pH value below 5.5. The addition of 0.03 M NaCl did not change the solubility pattern of either PI1 or PI2. However, the presence of 0.25 M NaCl diminished the solubility of the protein isolates for the entire pH area from 2 to 8.5. PI1 and PI2 may serve as a valuable source of sulfur-containing amino acids and arginine to complement human diets deficient in these specific amino acids.

Keywords Sunflower protein isolates Amino acids Solubility

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Introduction Sunflower meal is a by-product obtained after oil extraction from sunflower seeds. It contains relatively high amount of protein (30-50%) which may reach 66% depending on the efficiency of seed dehulling and defatting processes (Bau et al., 1983; Dorrell and Vick, 1997). Sunflower proteins are characterized with high nutritive value. They do not contain antinutritional compounds and exhibit well-balanced amino acid composition with the except for lysine which is the first limiting amino acid (Gassmann, 1983; González-Pérez and Vereijken, 2007). Sunflower proteins are rich in sulfur-containing amino acids which, in general, are deficient in most proteins with plant origin (Gassman, 1983; Ribarova et al., 1987; Canibe et al., 1999). Sunflower seeds contain 2 major protein groups, namely globulins and albumins. Sunflower albumins have low molecular weight and high solubility, which do not depend on pH and ionic strength of the solutions. The globulins, known as helianthinin, consist of oligomers with molecular weight from 300 to 350 kDa (11S globulins) which may dissociate to either oligomers with lower molecular weight (7S) or monomers (2S-3S). The dissociation and association of the helianthinin depends on pH, temperature and ionic strength of the solution (Sripad and Narasinga Rao, 1987; González-Pérez et al., 2004). At low ionic strength, water solubility of the globulins is at *Corresponding author. Email: [email protected] Tel: 0395 32 603 855

minimum at their isoelectric points ranging from pH 4.0 to pH 6.0. At high ionic strength of the solution, the helianthinin is almost insoluble in acidic medium (Canella et al., 1985; Rossi et al., 1985; Vermeesch et al., 1987; González-Pérez et al., 2004, 2008). The amino acid composition and solubility of the proteins in the sunflower meal, remaining after oil extraction, may vary and depend on the pre-treatment of the sunflower seeds and the procedure used for the oil production. Elevated temperature, which is frequently used to increase oil yield, favors protein denaturation and the formation of insoluble protein fraction (Lusas, 1985). In addition, it facilitates the protein interaction with other plant components leading to dark colored products which worsen the functional properties of the proteins (Parrado et al., 1993; Moure et al., 2006; González-Pérez and Vereijken, 2008; Salgado et al., 2011). Most of the studies related to sunflower protein isolates have been performed after defattining of the seeds under mild laboratory conditions where no change of the native structure and functionality of the proteins occurred. However, the industrial procedure of oil extraction from sunflower seeds may affect the amino acid composition, solubility and functional properties of the respective proteins. The protein isolates from industrially obtained sunflower meal, however, are of a higher practical interest since it is an alternative approach for a better and more complete use of this by-product for food application. For the

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purpose of their practical utilization, it is important to know the conditions which may affect the functional properties of the proteins. The purpose of this research was to establish the amino acid composition and water solubility of protein isolates, which were prepared from industrially produced sunflower meal, as a function of pH and NaCl concentrations. It was a continuation of a previous study where the optimal conditions for the extraction of the proteins from the same protein source have been established (Ivanova et al., 2011, 2012). Materials and Methods Preparation of protein isolates Protein isolates were obtained from sunflower meal, provided by a local oil factory, after extraction with 10% NaCl as described by Ivanova et al. (2011, 2012). Protein isolate 1 (PI1) was prepared by isoelectric precipitation of the extracted proteins. After adjusting pH to 2.5, the protein precipitate was collected by centrifugation (6000 rpm), washed twice with HCl (pH 2.5) and dried by lyophilization. Protein isolate 2 (PI2) was obtained by addition of 96% ethanol to the sunflower protein extract. The precipitate was collected by centrifugation (6000 rpm), washed twice with 96% ethanol and dried at 40°C. Amino acid analysis Amino acid composition of protein isolates was determined by using an amino acid analyzer (T 339 M, Praha Czechoslovakia) after acidic hydrolysis with 6 N HCI at 105°С for 24 h. To determine sulfurcontaining amino acids, the acidic hydrolysis of the protein isolate samples was preceded by a treatment with H2O2 and formic acid at 4°С for 12 h ( BNS 11374, 1986; Amarakoon, 2012). Amino acid score was calculated as a ratio of the amount of each еssential amino acid determined in a protein isolate and the amount of the respective amino acid in an “ideal” protein as stated by the Food and Agriculture Organization of the United Nations (FAO) and expressed in percent (FAO, 1970). Solubility of protein isolates as a function of pH and NaCl concentrations Solubility of the protein isolates was determined as described by González-Pérez (2003) with some modifications. Proteins were dispersed in water to a final concentration of 4 mg/ml. Variations in NaCl concentrations were achieved by adding NaCl. The influence of pH on protein solubility was tested by varying pH from 2 to 8.5 with an increment of 0.5 by

using NaOH or HCl. After 2 h at room temperature, samples were centrifuged (6 000 rpm) for 15 min and supernatants were collected. Protein solubility was calculated as a ratio of the amount of the protein in a supernatant as determined by Biuret method (AACC, 1983) and the initial concentration of the protein used in the test system (4 mg/ml). The result was multiplied by 100 to express in percent. Complete solubility (100%) was assumed when no residue was observed after centrifugation. All experiments were performed in triplicates. Each datum on Figures represents an average mean of the results generated from three independent experiments ± standard deviations. Results and Discussion Amino acid composition of protein isolates Numerous methods for preparation of protein isolates have been published. They include but are not limited to utilization of organic solvents, salts or/and reducing agents, and isoelectric precipitation (Rahma et al., 1981; Saeed et al., 1988; Pickardt et al., 2009). Ethanol and isoelctric precipitation were chosen to prepare the sunflower protein isolates because of their potential practical application and advantages related to protein recovery and reduced protein denaturation (Fan et al., 1976; Taha et al., 1981; Hewedi et al., 1985). Amino acid composition of protein isolates is a measure of their nutritive value. The concentrations of the amino acids in PI1 and PI2 did not differ from each other considerably (Table 1). It implies that the two approaches, namely ethanol and isoelectric precipitation, which were used for the preparation of the protein isolates, did not affect the amino acid profiles of the proteins. The amino acid contents of both protein isolates were comparable to those in the sunflower meal, used as the protein source in the study, with the except for lysine (Table 1, Ivanova et al., 2012). The concentrations of lysine in PI1 and PI2 did not exceed 1.97 g/100 g and 2.37 g/100 g protein respectively (Table 1), while the amount of this amino acid in the sunflower meal was determined to be 3.55 g/100 g protein (Ivanova et al., 2012). The decrease of the lysine amounts in the protein isolates is probably due to the interaction of the respective amino acid with other plant components during oil processing. As a result, lysine radicals form complex compounds which make the corresponding proteins insoluble in salt solution and slightly acidic medium which were used for the preparation of the protein isolates in our study. San Juan and Villamide (2001) who investigated the nutritional value of sunflower by-products also observed that lysine was the amino


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Table 1. Amino acid composition of sunflower protein isolates Amino a cids

“Idea l” protein* Amino a cid, g/100 g protein

Lys Met+Cys Thr Ile Leu Phe+Tyr Va l Trp

5.5 3.5 4.0 4.0 7.0 6.0 5.0 1.0

Asp Ser Glu Pro Gly Ala His Arg

Protein isola te 1 Protein isola te 2 Amino a cid, Amino a cid Amino a cid, Amino a cid g/100 g protein score, % g/100 g protein score, % Essentia l a mino a cids 1.97 35.81 2.37 43.09 3.47 99.14 3.47 99.14 3.39 84.75 3.13 78.25 3.16 79.00 2.61 65.25 6.18 88.29 5.64 80.57 8.56 142.67 7.86 131.00 3.58 71.60 3.16 63.20 ND** ND** Nonessentia l a mino a cids 10.50 9.76 4.50 5.33 26.91 25.39 5.11 5.65 4.88 8.34 4.98 5.46 3.06 3.38 9.75 8.45

Amino acid composition of an “ideal” protein as published by FAO (1970) Not determined

*

**

acid to be affected by sunflower oil processing the most. Valine and isoleucine appeared to be the second and third limiting amino acids in our protein isolates as the decreases in the amino acid scores for PI2 (63.20% and 65.25%) was more prominent than for PI1 (71.60% and 79.00%) when compared to the respective amino acids in the sunflower seeds (115.40% and 119.25%) (Ribarova et al., 1987). Despite of the deficiency of lysine, valine and isoleucine, PI1 and PI2 have the potential to be used in food industry because of their relatively high amount of sulfur-containing amino acids (Table 1). Sulfurcontaining amino acids i.e. methionine and cystine limit the nutritive value of numerous plant proteins. A study on protein and amino acid composition of wild and cultivated genotypes of Phaseolus species by Baudoin and Maquet (1999) implied that both plant protein sources should be mixed with other plant species which are rich in methionine and cystine to overcome the deficiency of these amino acids. According to Burstin et al. (2011), legume seeds such as pea, fababean, lentil and cowpea, which are commonly used in human nutrition, should be combined with other plant species to balance the diet with respect to methionine and cystine. In addition, both protein isolates exhibited relatively high amounts of arginine which in PI1 and PI2 reached 9.75 and 8.45 g/100 g protein respectively (Table 1). Arginine is considered a semi-essential amino acid for humans and participates in numerous biochemical processes such as ammonia detoxification, hormone secretion, and immune modulation (Appleton, 2002). Humans can synthesize it de novo but there is no compensatory mechanism in case of depletion and dietary supplementation appears to be the way to replenish the plasma concentrations of this amino acid (Castillo et al., 1994). Although containing

relatively small amounts of histidine, PI1 (3.06 g/100 g protein) and PI2 (3.38 g/100 g protein) may also potentially serve as a source of this amino acid which is considered essential for children. If compared to data on amino acids in Bulgarian food products which were investigated by Ribarova et al. (1987), the levels of arginine and histidine in PI1 (9.75 and 3.06 g/100 g protein) and PI2 (8.45 and 3.38 g/100 g protein) exceeded the respective amino acids in egg protein (6.09 and 2.4 g/100 g protein) and milk (3.28 and 2.45 g/100 g protein). Solubility of PI1 and PI2 as a function of pH and NaCl concentrations Solubility of proteins depends on numerous factors such as amino acid composition, pH, presence of salts, interaction with other matrix components and denaturation (Kinsela et al., 1985). PI1 and PI2 did not differ substantially in their amino acid composition (Table 1). Therefore, the amino acid profile of both isolates would not be expected to be a dominant factor influencing their solubility. According to Damodaran (1994), the solubility of proteins is highly dependent on their conformation. By studying the conformational states of sunflower proteins, González-Pérez et al. (2004) demonstrated that helianthinin conformation was modulated by pH, salt concentrations and thermal denaturation. The influence of pH on the solubility of PI1 and PI2 is presented on Figure 1. Both isolates exhibited similar pattern of solubility at pH ≥ 4. PI1 was insoluble in pH ranging from 4.0 to 5.5. Out of this range, the solubility gradually increased by reaching maximum values at pH 7 and pH 2. The solubility pattern exhibited by PI1 as a function of pH is similar to the solubility of helianthinin and other sunflower protein isolates at low ionic strength (Rossi et al.,


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100

100

80

Solubility, %

Solubility, %

80 60 40

60 40 20

20

0 0

0 2

3

4

5

6

7

8

0.05

0.1

0.15

pH 7

рН PI 1

PI 2

Figure 1. Influence of pH on solubility of protein isolate 1 (PI1) and protein isolate 2 (PI2)

0.2

0.25

NaCl conce ntracions (M)

9

pH3

Figure 3. Solubility of protein isolate 1 as a function of different NaCl concentrations at pH 3 and pH 7 100 80

Solubility, %

100

Solubility, %

80 60

60 40 20 0

40

2

3

4

5

6

7

8

9

рН

20

0.03 M NaCl

0 2

3

4

5

6

7

8

9

рН 0.03 M NaCl

0.25 M NaCl

Figure 2. Influence of pH on solubility of protein isolate 1 in the presence of different concentrations of NaCl

1985; Vermeesch et al., 1987; Saeed and Cheryan, 1988; González-Pérez, 2003; González-Pérez et al., 2004). PI2 was almost insoluble at pH ≤ 5.5 but reached its complete solubility at pH ≥ 7. Similar behavior of solubility of sunflower protein isolates was observed by González-Pérez (2003) when 250 mM NaCl were added to protein suspensions. The data on PI1 and PI2 solubility presented on Figure 1 imply that in addition to pH other factor(s) which modulate their solubility may exist. The differences in solubility pattern of PI1 and PI2 are probably due to their different chemical composition. PI1 was characterized with higher protein content (94.25%) than PI2 (75.34%) (Ivanova et al., 2011). However, PI2 exhibited approximately 10-fold higher ash content (13.26%) than PI1 (1.34%) which probably influenced the lower solubility of this isolate at pH ≤ 5.5. Numerous studies have been published on sunflower proteins. However, most of them are related to undenatured helianthinin and protein isolates (Canella, 1985; Vermeesch, 1987; González-Pérez, 2003). Since industrially produced sunflower meal was used as a protein source in our study, it could be expected that the isolated proteins were pre-denatured at some extend and would have different solubility pattern in salt solutions when compared to undenatured proteins. Two levels of salt concentrations (0.03 and 0.25 M NaCl) were used to investigate the solubility of PI1 and PI2 in the presernce of salts. No change in

0.25 M NaCl

Figure 4. Influence of pH on solubility of protein isolate 2 in the presence of different concentrations of NaCl

the solubility pattern of PI1 after the addition of 0.03 M NaCl occurred (Figure 2). However, the solubility of PI1 highly decreased in the presence of 0.25 M NaCl and reached only 55% of its capacity at pH 8.5. The isolate remained insoluble at each pH value lesser than 6. Since the most profound modulation in the solubility of PI1 occurred at pH 3 and pH 7, it was investigated in more details for both pH values in the presence of NaCl which concentrations were varied from 0 to 0.25 M with an increment of 0.05 (Figure 3). The results indicated that relatively low concentrations of NaCl limited the solubility of PI1 as more noticeable changes occurred in the range from 0.05 to 0.15 M NaCl. At 0.15 M NaCl, PI1 exhibited 30% solubility in neutral medium (pH 7) and remained insoluble at pH 3. In contrast, at the same salt concentration, undenatured protein isolates expressed 80% solubility at pH 3 and remained as high as 90% soluble at pH 7 even when higher concentrations of NaCl were added (González-Pérez, 2003). The addition of 0.03 M NaCl did not change substantially the solubility profile of PI2 (Figure 1 and Figure 4). However, the supplementation of the solution with the higher level of NaCl (0.25 M) diminished the maximum solubility of the isolate with approximately 25%. As a whole, the solubility of both protein isolates was influenced by the addition of NaCl. Even relatively small amounts of salts decreased the solubility of PI1 at pH 3 as well as pH 7. It is probably due to the partial denaturation of the proteins in the sunflower meal that was used for the preparation of the protein isolates. Elevated temperature used during


oil extraction and the production of sunflower meal probably exerted denaturation effect on sunflower seed proteins. Most probably, lysine radicals from the proteins of the sunflower meal interacted with other seed components thus diminishing the positive charge of the protein molecules in acidic medium. Therefore, only small amounts of chloride anions would be sufficient to neutralize the positive charge of the protein molecules after which the latter become capable in aggregating and coagulating. In alkaline medium, the proteins are charged negatively and cations have low affinity to bind (Schnepf, 1992). Thus, salts appear to be less powerful modulator of protein solubility in alkaline medium. Conclusions In summary, PI1 and PI2 differed from each other in their solubility profiles, which was probably due to the differences in their chemical contents. The addition of 0.03 M NaCl did not change the solubility pattern of either PI1 or PI2. However, 0.25 M NaCl decreased the solubility of both protein isolates in the entire pH scale ranging from 2 to 8.5. Our study indicated that PI1 and PI2 could be potentially used as valuable additives in food industry due to their amino acid composition. They could supplement human diet with sulfur-containing essential amino acids which, in general, are deficient in most proteins with plant origin. The relatively high amounts of histidine and arginine additionally increased the nutritive value of PI1 and PI2. References AACC. 1983. Method 46-15: Crude protein – 5-minute biuret method for wheat and other grains. Approved Method of American Association of Cereal Chemists, St. Paul, MN, USA. Amarakoon, R. 2012. Study on amino acid content in selected varieties of Pisum sativum (peas) by ion exchange chromatography. International Conference on Nutrition and Food Sciences (IPCBEE), v. 39. Singapore: IACSIT Press. Appleton, J. 2002. Arginine: Clinical potential of semiessential amino acid. Alternative Medical Review 7(6): 512-522. Bau, H. M., Mohtadi Nia, D. J., Mejean, L. and Debry, G. 1983. Preparation of colorless sunflower protein products: Effect of processing on physicochemical and nutritional properties. Journal of the American Oil Chemists’ Society 60: 1141-1148. Baudoin, J. P. and Maquet, A. 1999. Improvement of protein and amino acid contents in seeds of food legumes. A case study in Phaseolus. Biotechnology, Agronomy, Society and Environment 3 (4): 220–224..

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Bulgarian National Standard (BNS) 11374. 1986. Determination of amino acids in complete feeds, protein ingredients, and sources for them after hydrolysis by using chemical and microbiological methods. p. 50. Burstin, J., Gallardo, K., Mir, R. R., Varshney, R. K. and Duc, G. 2011. Improving protein content and nutrition quality. In Biology and Breeding of Food Legumes, p. 314-328. Wallingford: CAB International Canella, M., Castriotta, G., Bernardi, A. and Boni, R. 1985. Functional properties of individual sunflower albumin and globulin. Lebensmittel Wissenschaft & Technologie 18: 288-292. Canibe, N., Pedrosa, M. M., Robredo, L. M. and Knudsen, K. E. B. 1999. Chemical composition, digestibility and protein quality of 12 sunflower (Helianthus annuus L.) cultivars. Journal of the Science of Food and Agriculture 79 (13): 1775-1782. Castillo, L., Ajami, A., Branch, S., Chapman, T. E., Yu, Y. M., Burke, J. F. and Young, V. R. 1994. Plasma arginine kinetics in adult man: response to an argininefree diet. Metabolism 43: 114-122. Damodaran, S. 1994. Structure-function relationship of food protein, In Hettiarachchy, N. S. and Ziegler, G. R. (Eds). Protein Functionality in Food Systems, p. 1-38, New York: Marcel Dekker Inc. Dorrell, D.G. and Vick, B.A. 1997. Properties and processing of oilseed sunflower. In Schneiter, A. A. (Ed.). Sunflower Technology and Production, p. 709744. Madison, WI: American Society of Agronomy, Crop Science Society of America, Soil Science Society of America. Fan, T. Y. and Sosulski, F. W. 1976. New techniques for preparation of improved sunflower protein concentrates. Cereal Chemistry 53(1): 118-125. FAO. 1970. Nutritional Studies No24 Amino acid content of foods and biological data on proteins. FAO, Rome. Gassmann, B. 1983. Preparation and application of vegetable proteins, especially proteins from sunflower seed, for human consumption. An approach. Nahrung 27: 351-369. González-Pérez, S. 2003. Physico-chemical and functional properties of sunflower proteins. Wageningen, The Netherlands: Wageningen University, PhD dissertation. González-Pérez, S., Vereijken, J. M., Merck, K. B., van Koningsveld, G.. A., Gruppen, H. and Voragen, A., G. 2004. Comformational stats of sunflower (Helianthus annuus) helianthinin: effect of heat and pH. Journal of Agriculture and Food Chemistry 53(22): 6770-6778. González-Pérez, S. and Vereijken, J. M. 2007. Sunflower proteins: overview of their physicochemical, structural and functional properties. Journal of the Science of Food and Agriculture 87: 2172-2191. Hewedi, M. M., Mulvihill, D. M. and Fox, P. F. 1985. Recovery of milk protein by ethanol precipitation. Irish Journal of Food Science and Technology 9(1): 11-23. Ivanova, P., Koleva, L., Tziporkov, N. and Pishtiyski, I. 2011. Isolation of proteins from salt extracts of

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sunflower meal. SW UFT-Plovdiv LVIII, 2: 391-396. Ivanova, P., Chalova, V., Koleva, L., Pishtiyski, I. and Perifanova-Nemska, M. 2012. Optimization of protein extraction from sunflower meal produced in Bulgaria. Bulgarian Journal of Agricultural Science 18(2): 153160. Kinsella, J. E., Damodaran, S. and German, B. 1985. Physicochemical and functional properties of oilseed proteins with emphasis on soy proteins. New Protein Foods 5: 107–179. Lusas, E. W. 1985. Sunflowerseed protein. In Altschul, A. M. and Wilcke, H. L. (Eds).. New Protein Foods, Volume 5, Seed Storage Proteins, p. 393-433. New York: ,Academic Press. Moure, A., Sineiro, J., Dominguez, H. and Parajo, J. C. 2006. Functionality of oilseed protein products: a review. Food Res International 39: 945-963. Parrado, J., Millan, F., Hernandez-Pinzon, I., Bautista, J. and Machado, A. 1993. Characterization of enzymatic sunflower protein hydrolysates. Journal of Agriculture and Food Chemistry 41: 1821-1825. Pickardt, C., Neidhart, S., Griesbach, C., Dube, M., Knauf, U., Kammerer, D. and Carle, R. 2009. Optimisation of mild-acidic protein extraction from defatted sunflower (Helianthus annuus L.) meal. Food Hydrocolloids 23: 1966-1973. Rahma, E. H. and Narasinga Rao, M. S. 1981. Isolation and characterization of the major protein fraction of sunflower seeds. Journal of Agriculture and Food Chemistry 29: 518-521. Ribarova, F., Shishkov, S. and Baklova, I. 1987. Amino acid composition of Bulgarian food products. Sofia, Bulgaria: Zemizdat. Rossi, M., Pagliarini, E. and Peri, C. 1985. Emulsifying and foaming properties of sunflower protein derivatives. Lebensmittel Wissenschaft & Technologie 18: 293299. Saeed, M. and Cheryan, M. 1988. Sunflower protein concentrates and isolates low in polyphenols and phytate. Journal of Food Science 53(4): 1127-1132. Salgado, P. R., Molina Ortiz, S. E., Petruccelli, S. and Mauri, A. N. 2001. Sunflower protein concentrates and isolates prepared from oil cakes have high water solubility and antioxidant capacity. Journal of the American Oil Chemists’ Society 88: 351-360. San Juan, L. D. and Villamide, M. J. 2001. Nutritional evaluation of sunflower products for poultry as affected by the oil extraction process. Poultry Science 80: 431–437. Schnepf, M. I. 1992. Protein-water interactions. In Hudson B. J. F (Ed) Biochemistry of Food Proteins, p. 1-33. Springer US Sripad, G. and Narasinga Rao, M. S. 1987. Effect of acid pH on the 11S protein of sunflower seed. Journal of Agriculture and Food Chemistry 35: 668-672. Taha, F. S., Abbasy, M., El-Nockrashy, A. S. and Zein, E. 1981. Countercurrent extraction-isoelectric precipitation of sunflower seed protein isolates. Journal of the Science of Food and Agriculture 32(2): 166-174.

Vermeesch, G., Briffaud, J. and Joyeux, J. 1987. Sunflower proteins in human food. Revue Francaise des Corps Gras 7-8: 333-344.